TWI729290B - Pharmaceutical compositions for treating pain - Google Patents

Pharmaceutical compositions for treating pain Download PDF

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TWI729290B
TWI729290B TW107116441A TW107116441A TWI729290B TW I729290 B TWI729290 B TW I729290B TW 107116441 A TW107116441 A TW 107116441A TW 107116441 A TW107116441 A TW 107116441A TW I729290 B TWI729290 B TW I729290B
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morphine
pain
mice
pharmaceutical composition
tolerance
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敏 卓
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香港商永展國際有限公司
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Abstract

The present invention discloses a novel use of a pharmaceutical composition in the preparation of a medicament in the treatment of opioid-induced tolerance and addiction, in particular to the use of AC1 inhibitor NB001 and AC1&8 mixed inhibitors NB010 and NB011 in the preparation of a medicament in the treatment of opioid-induced tolerance and addiction.

Description

治療疼痛的藥物組合物Pharmaceutical composition for treating pain

本發明涉及一種藥物組合物在製備治療由類鴉片誘導的耐受性和成癮的藥物中的新用途，具體涉及AC1抑制劑NB001以及AC1＆8混合抑制劑NB010和NB011在製備治療由類鴉片導致的耐受性和成癮的藥物中的應用。The present invention relates to a new use of a pharmaceutical composition in the preparation of drugs for the treatment of tolerance and addiction induced by opioids, in particular to the preparation of AC1 inhibitor NB001 and AC1&8 mixed inhibitors NB010 and NB011 in the preparation and treatment of opioids induced Tolerance and application of addictive drugs.

術語“鴉片製劑(opiate)”已經被用於指定衍生自鴉片(opium)的藥理活性生物鹼類，如嗎啡、可待因、和許多嗎啡的半合成同類物。在分離出具有嗎啡樣作用的肽化合物後，導入術語類鴉片(opioid)，其一般指具有嗎啡樣作用的所有藥物。在類鴉片中，包括表現嗎啡樣活性的各種肽，如內啡肽、腦啡肽、和強啡肽。然而，某些原始資料使用一般意義的術語“鴉片製劑”，在這樣的情況下，鴉片製劑與類鴉片可互換。此外，術語類鴉片已經被用於指嗎啡樣藥物的拮抗劑，並用於鑑定與此類藥物組合的受體或結合位元點。The term "opiate" has been used to designate pharmacologically active alkaloids derived from opium, such as morphine, codeine, and many semi-synthetic congeners of morphine. After isolating peptide compounds with morphine-like effects, the term opioid was introduced, which generally refers to all drugs with morphine-like effects. The opioids include various peptides that exhibit morphine-like activity, such as endorphins, enkephalins, and dynorphins. However, some sources use the generic term "opiate", in which case opiates and opioids are interchangeable. In addition, the term opioid has been used to refer to antagonists of morphine-like drugs and to identify receptors or binding sites in combination with such drugs.

雖然類鴉片通常用作鎮痛藥，嗎啡常用於緩解一些劇痛，然而嗎啡的臨床應用性受限於鎮痛耐受性、身體依賴性以及成癮的發展。已知長期服用嗎啡可誘導出人和動物的耐受性，但是不能對μ-受體脫敏。相反，長期使用嗎啡會導致腺苷酸環化酶（AC）的過敏作用（hypersensitization），這會助長類鴉片耐受性和依賴性(Nestler, 1997; 2001a,b; 2002)。根據文獻，在幾個腦區域中的腺苷酸環化酶活性的長期變化，例如中腦腹側被蓋區（VTA）和伏隔核（NAc）區域（兩者對於鴉片藥物強化都很關鍵），以及在藍斑和背側核（對於鴉片戒斷比較關鍵）（Nestler和Aghajanian，1997; Jolas等，2000; Williams等，2001; Chao和Nestler，2004）。Although opioids are commonly used as analgesics and morphine is often used to relieve some severe pain, the clinical applicability of morphine is limited by the development of analgesic tolerance, physical dependence and addiction. It is known that long-term use of morphine can induce tolerance in humans and animals, but it cannot desensitize μ-receptors. Conversely, long-term use of morphine can cause hypersensitization of adenylate cyclase (AC), which promotes opioid tolerance and dependence (Nestler, 1997; 2001a,b; 2002). According to the literature, long-term changes in adenylate cyclase activity in several brain regions, such as the midbrain ventral tegmental area (VTA) and the nucleus accumbens (NAc) region (both are critical for opioid enhancement ), and in the locus coeruleus and the dorsal nucleus (which are critical for opium withdrawal) (Nestler and Aghajanian, 1997; Jolas et al., 2000; Williams et al., 2001; Chao and Nestler, 2004).

儘管提示了ACs可調節類鴉片類物質的作用，但是由於缺少特異性抑制劑來定義不同的AC同工酶的作用，導致了研究進展較為緩慢。迄今，10種ACs的基因已經被克隆，每個在中樞神經系統和外周感覺神經系統中具有不同的表達模式（Xia和Storm，1997）。其中，AC1和AC8僅由腦中受Ca2+/鈣調蛋白刺激。 AC1和AC8廣泛分佈在不同的大腦區域，包括VTA、NAc、藍斑、和中縫背核（Zhuo，2012）。Although it is suggested that ACs can modulate the role of opioids, the lack of specific inhibitors to define the role of different AC isoenzymes has led to slower progress in research. So far, 10 ACs genes have been cloned, each with a different expression pattern in the central nervous system and peripheral sensory nervous system (Xia and Storm, 1997). Among them, AC1 and AC8 are only stimulated by Ca2+/calmodulin in the brain. AC1 and AC8 are widely distributed in different brain regions, including VTA, NAc, locus coeruleus, and dorsal raphe nucleus (Zhuo, 2012).

此外，有許多研究涉及藥物成癮中的CREB（Guitart等，1992; Hyman，1996; Maldonado等，1996; Walters等，2001）。 西方墨點法的分析顯示嗎啡耐受動物中CREB或磷酸化CREB（pCREB）的表達增加（Li等，1999; Gao et al。，2004），CREB突變小鼠在停止嗎啡治療後顯示較不嚴重的戒斷症狀（ Maldonado等，1996）。另外，主要介導嗎啡生理作用的G蛋白偶聯受體M類鴉片受體（M opioid receptor ，MOR）包含CRE元素，並被證明通過CREB介導的途徑被啟動（Min et al。，1994; Nestler ，1997; Lee等，2003）。在中樞神經系統中，已知AC1和AC8作用於上游的關鍵信號蛋白，來調節神經元中的CREB（Wei等。，2002; Zhuo，2012）。通過使用AC1和AC8單基因剔除（KO）小鼠、以及DKO小鼠，據報導，AC1或AC8基因剔除小鼠、或DKO小鼠與野生型小鼠在短期內對嗎啡的鎮痛效應中沒有差異（Li等，2006）。AC1&8 DKO 小鼠以及AC8單基因剔除小鼠，通過每日注射嗎啡（10mg/kg體重）持續誘導的耐受性降低。在持續用嗎啡治療的DKO小鼠中，戒斷行為顯著降低（Li 等，2006）。最近使用AC1抑制劑ST034037研究發現，AC1活性確實需要MOR慢性活化誘導的AC致敏，這表明AC1抑制可能有益於克服慢性嗎啡誘導的耐受性和其他反應（Brust等，2017）。In addition, there are many studies involving CREB in drug addiction (Guitart et al., 1992; Hyman, 1996; Maldonado et al., 1996; Walters et al., 2001). Western blot analysis showed that CREB or phosphorylated CREB (pCREB) expression increased in morphine-tolerant animals (Li et al., 1999; Gao et al., 2004), and CREB mutant mice showed less severe symptoms after stopping morphine treatment Withdrawal symptoms (Maldonado et al., 1996). In addition, the G protein-coupled receptor (M opioid receptor, MOR), which mainly mediates the physiological effects of morphine, contains CRE elements and has been proven to be activated through CREB-mediated pathways (Min et al., 1994; Nestler, 1997; Lee et al., 2003). In the central nervous system, AC1 and AC8 are known to act on key upstream signaling proteins to regulate CREB in neurons (Wei et al., 2002; Zhuo, 2012). By using AC1 and AC8 single gene knockout (KO) mice and DKO mice, it is reported that AC1 or AC8 knockout mice, or DKO mice and wild-type mice have no difference in the analgesic effect of morphine in the short term (Li et al., 2006). AC1&8 DKO mice and AC8 single-gene knock-out mice continuously induced decreased tolerance through daily injection of morphine (10 mg/kg body weight). In DKO mice continuously treated with morphine, withdrawal behavior was significantly reduced (Li et al., 2006). A recent study using the AC1 inhibitor ST034037 found that AC1 activity does require AC sensitization induced by chronic activation of MOR, indicating that AC1 inhibition may be beneficial to overcome chronic morphine-induced tolerance and other reactions (Brust et al., 2017).

在使用類鴉片類藥物治療疼痛過程中，療效的下降往往以藥理學上的耐受機制來解釋（或原有病情的加重），必須加大劑量才能維持原有療效。最近的研究證實，類鴉片類藥物除了產生鎮痛作用之外，還能夠啟動體內的促傷害機制，導致機體對疼痛的敏感性增高，即類鴉片誘發的痛覺過敏(opioid-induced hyperalgesia，OIH)。早先在類鴉片戒斷反應中就觀察到有疼痛敏感性出現，最新的證據表明，疼痛敏感性亦可發生於類鴉片治療過程中。也就是說，停用類鴉片和持續給予類鴉片都會造成疼痛敏感性增加，這顯然是矛盾的。因此，類鴉片鎮痛效果的降低很可能是 OIH（促傷害機制）的作用，而不僅僅是藥理學上的耐受機制。其中，既有敏化過程參與，也有去敏化過程發生。OIH有其獨特的細胞機制，涉及內源性強啡肽、谷氨酸能系統以及下行易化機制。有趣的是，OIH 的機制與神經病理痛以及類鴉片耐受的機制有很大程度的交疊。例如，外周神經損傷和重複類鴉片給藥能夠啟動相同的谷氨酸能細胞通路。In the process of using opioids to treat pain, the decrease in efficacy is often explained by the pharmacological tolerance mechanism (or the aggravation of the original condition), and the original effect must be increased to maintain the original effect. Recent studies have confirmed that, in addition to producing analgesic effects, opioids can also initiate injury-promoting mechanisms in the body, leading to increased body sensitivity to pain, that is, opioid-induced hyperalgesia (OIH). Earlier, pain sensitivity has been observed in opioid withdrawal. The latest evidence shows that pain sensitivity can also occur during opioid treatment. In other words, stopping opioids and continued administration of opioids will cause increased pain sensitivity, which is obviously contradictory. Therefore, the reduction in the analgesic effect of opioids is likely to be the effect of OIH (injury mechanism), not just a pharmacological tolerance mechanism. Among them, both the sensitization process participates and the desensitization process takes place. OIH has its own unique cellular mechanism, involving endogenous dynorphin, glutamatergic system and downward facilitating mechanism. Interestingly, the mechanism of OIH overlaps to a large extent with the mechanisms of neuropathic pain and opioid tolerance. For example, peripheral nerve injury and repeated opioid administration can initiate the same glutamatergic cell pathway.

使用類鴉片藥物治療過程中，藥物鎮痛效能的下降被認為是一種藥理學上的耐受現象，或者是現有疼痛狀態加重。因此，追加藥物劑量被理所應當地成為重建藥物作用的唯一選擇。但是根據臨床和實驗室研究所提供的有關類鴉片誘發疼痛的矛盾證據，發明人必須重新考慮對這一現象的解釋。一方面，如果類鴉片療效降低與藥物耐受機制有關，那麼加大用藥劑量顯然是無可非議的；而另一方面，倘若由於 OIH 所致，那麼盲目地增加劑量只會增強體內的促傷害過程，從而加劇疼痛和耐受。In the course of treatment with opioids, the decrease in the analgesic efficacy of the drug is considered to be a pharmacological tolerance phenomenon, or an aggravation of the existing pain state. Therefore, supplementing the drug dose is rightly the only option to rebuild the effect of the drug. However, based on the contradictory evidence about opioid-induced pain provided by clinical and laboratory research, the inventor must reconsider the explanation for this phenomenon. On the one hand, if the reduction in the efficacy of opioids is related to the drug tolerance mechanism, then increasing the dose is obviously beyond reproach; on the other hand, if it is caused by OIH, then blindly increasing the dose will only enhance the harm-promoting process in the body. This intensifies pain and tolerance.

因此為了延長類鴉片類藥物的止痛時效，克服臨床應用中的鎮痛耐受性、身體依賴性、以及成癮性，新的藥物或聯合制劑的研發具有重要的意義。Therefore, in order to prolong the analgesic effect of opioids and overcome the analgesic tolerance, physical dependence, and addiction in clinical applications, the development of new drugs or combination preparations is of great significance.

現有技術文獻Prior art literature

非專利文獻 Brust et al (2017) Identification of a selective small-molecule inhibitor of type 1 adenylyl cyclase activity with analgesic properties. Science signaling 10.Non-patent literature Brust et al (2017) Identification of a selective small-molecule inhibitor of type 1 adenylyl cyclase activity with analgesic properties. Science signaling 10.

專利文獻 美國專利公開號 US2011/0098295Patent Literature US Patent Publication No. US2011/0098295

鑑於上述類鴉片類藥物的鎮痛耐受性、身體依賴性、以及成癮性等問題，本發明的目的在於開發出一種新的藥物或聯合制劑，來減少或改善長期使用類鴉片類藥物而帶來的鎮痛耐受性、身體依賴性、以及成癮性等問題，即減少長期使用嗎啡和其他類鴉片類藥物引起的附加反應。In view of the analgesic tolerance, physical dependence, and addiction of the above-mentioned opioid drugs, the purpose of the present invention is to develop a new drug or combination preparation to reduce or improve the long-term use of opioid drugs. The problems of analgesia tolerance, physical dependence, and addiction are to reduce the additional reactions caused by long-term use of morphine and other opioids.

已知類鴉片可以啟動細胞中的cAMP產生，類鴉片的長期使用會導致腺苷醯基酶的過敏反應。並且cAMP依賴性信號通路對於類鴉片相關的長期作用包括耐受性、依賴性及其它因素中具有關鍵的影響（參見Li 等。，2006; Brust 等，2017）。It is known that opioids can initiate the production of cAMP in cells, and long-term use of opioids can cause allergic reactions to adenosylase. And the cAMP-dependent signaling pathway has a key influence on the long-term effects of opioids, including tolerance, dependence, and other factors (see Li et al., 2006; Brust et al., 2017).

為了完成上述發明，本發明人在研究過程中得知，本發明所揭的三種化合物NB001、NB010和NB011可用於減少長期使用類鴉片類藥物引起的附加反應，從而完成了本發明。 In order to complete the above-mentioned invention, the inventors learned during the research that the three compounds NB001, NB010 and NB011 disclosed in the present invention can be used to reduce the additional reactions caused by long-term use of opioids, thus completing the present invention.

本發明的NB001，即5-((2-(6-氨基-9H-嘌呤-9-基)乙基)胺基)戊烷-1-醇，具有以下結構：

Figure 107116441-A0305-02-0006-1
The NB001 of the present invention, namely 5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentane-1-ol, has the following structure:
Figure 107116441-A0305-02-0006-1

5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentan-1-ol 5-((2-(6-amino-9 H -purin-9-yl)ethyl)amino)pentan-1-ol

本發明的NB010，即6-氨基-9-(2-對-甲苯氧基-乙基)-9H-嘌呤-8-硫醇，具有以下化學結構：

Figure 107116441-A0305-02-0006-2
The NB010 of the present invention, namely 6-amino-9-(2-p-tolyloxy-ethyl)-9H-purine-8-thiol, has the following chemical structure:
Figure 107116441-A0305-02-0006-2

6-amino-9-(2-p-tolyloxy-ethyl)-9H-purine-8-thiol 6-amino-9-(2-p-tolyloxy-ethyl)-9H-purine-8-thiol

本發明的NB011，即4-(9H-嘌呤-6-基)嗎啉(也稱為6-嗎啉-4-基-7H-嘌呤)具有以下化學結構：

Figure 107116441-A0305-02-0007-3
The NB011 of the present invention, namely 4-(9H-purin-6-yl)morpholine (also known as 6-morpholin-4-yl-7H-purine) has the following chemical structure:
Figure 107116441-A0305-02-0007-3

6-morpholin-4-yl-7H-purine 6-morpholin-4-yl-7H-purine

根據本發明，提供了如下內容： According to the present invention, the following contents are provided:

(1)、一種治療疼痛的藥物組合物，其特徵在於含有：類鴉片類藥物，以及選自NB001、NB010、或NB011三種化合物中的至少1種，其中：所述NB001，即5-((2-(6-氨基-9H-嘌呤-9-基)乙基)胺基)戊烷-1-醇，具有以下結構：

Figure 107116441-A0305-02-0007-4
(1) A pharmaceutical composition for the treatment of pain, characterized in that it contains: opioids and at least one compound selected from the group consisting of NB001, NB010, or NB011, wherein: the NB001 is 5-(( 2-(6-Amino-9H-purin-9-yl)ethyl)amino)pentane-1-ol has the following structure:
Figure 107116441-A0305-02-0007-4

5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentan-1-ol 5-((2-(6-amino-9 H -purin-9-yl)ethyl)amino)pentan-1-ol

所述NB010，即6-氨基-9-(2-對-甲苯氧基-乙基)-9H-嘌呤-8-硫醇，具有以下化學結構：

Figure 107116441-A0305-02-0007-5
The NB010, namely 6-amino-9-(2-p-tolyloxy-ethyl)-9H-purine-8-thiol, has the following chemical structure:
Figure 107116441-A0305-02-0007-5

6-amino-9-(2-p-tolyloxy-ethyl)-9H-purine-8-thiol 6-amino-9-(2-p-tolyloxy-ethyl)-9H-purine-8-thiol

所述NB011，即4-(9H-嘌呤-6-基)嗎啉(也稱為6-嗎啉-4-基-7H-嘌呤)具有以下化學結構：

Figure 107116441-A0305-02-0008-6
The NB011, namely 4-(9H-purin-6-yl)morpholine (also known as 6-morpholin-4-yl-7H-purine) has the following chemical structure:
Figure 107116441-A0305-02-0008-6

6-morpholin-4-yl-7H-purine。 6-morpholin-4-yl-7H-purine.

(2)、如(1)所述的藥物組合物，其特徵在於，所述類鴉片類藥物是嗎啡。 (2) The pharmaceutical composition according to (1), wherein the opioid drug is morphine.

(3)、如(1)所述的藥物組合物，其特徵在於，所述NB001、NB010、或NB011三種化合物還包括其可藥用的鹽或溶劑合物。 (3) The pharmaceutical composition as described in (1), characterized in that the three compounds of NB001, NB010, or NB011 further include their pharmaceutically acceptable salts or solvates.

(4)、一種藥物組合物在製備治療疼痛藥物中的應用，所述藥物組合物含有：類鴉片類藥物，以及選自NB001、NB010、或NB011三種化合物中的至少1種，其中：所述NB001，即5-((2-(6-氨基-9H-嘌呤-9-基)乙基)胺基)戊烷-1-醇，具有以下結構：

Figure 107116441-A0305-02-0008-7
(4) An application of a pharmaceutical composition in the preparation of a pain medicine, the pharmaceutical composition containing: an opioid drug and at least one compound selected from the group consisting of NB001, NB010, or NB011, wherein: the NB001, namely 5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentane-1-ol, has the following structure:
Figure 107116441-A0305-02-0008-7

5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentan-1-ol 5-((2-(6-amino-9 H -purin-9-yl)ethyl)amino)pentan-1-ol

所述NB010，即6-氨基-9-(2-對-甲苯氧基-乙基)-9H-嘌呤-8-硫醇，具有以下化學結構：

Figure 107116441-A0305-02-0009-8
The NB010, namely 6-amino-9-(2-p-tolyloxy-ethyl)-9H-purine-8-thiol, has the following chemical structure:
Figure 107116441-A0305-02-0009-8

6-amino-9-(2-p-tolyloxy-ethyl)-9H-purine-8-thiol 6-amino-9-(2-p-tolyloxy-ethyl)-9H-purine-8-thiol

所述NB011，即4-(9H-嘌呤-6-基)嗎啉(也稱為6-嗎啉-4-基-7H-嘌呤)具有以下化學結構：

Figure 107116441-A0305-02-0009-9
The NB011, namely 4-(9H-purin-6-yl)morpholine (also known as 6-morpholin-4-yl-7H-purine) has the following chemical structure:
Figure 107116441-A0305-02-0009-9

6-morpholin-4-yl-7H-purine。 6-morpholin-4-yl-7H-purine.

(5)、如(4)所述的藥物組合物，其特徵在於，所述類鴉片類藥物是嗎啡。 (5) The pharmaceutical composition according to (4), wherein the opioid drug is morphine.

(6)、如(4)所述的藥物組合物，其特徵在於，所述NB001、NB010、或NB011三種化合物還包括其可藥用的鹽或溶劑合物。 (6) The pharmaceutical composition according to (4), characterized in that the three compounds of NB001, NB010, or NB011 further include their pharmaceutically acceptable salts or solvates.

(7)、一種治療疼痛的方法，其特徵在於：在使用類鴉片類藥物的基礎上，聯合使用至少1種選擇由NB001、NB010、或NB011三種化合物，及其可藥用的鹽或溶劑合物構成的群組，其中： 所述NB001，即5-((2-(6-氨基-9H-嘌呤-9-基)乙基)胺基)戊烷-1-醇，具有以下結構：

Figure 107116441-A0305-02-0010-10
(7) A method for the treatment of pain, characterized in that: on the basis of the use of opioids, at least one compound selected from NB001, NB010, or NB011, and its pharmaceutically acceptable salts or solvates are used in combination. Among them: the NB001, namely 5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentane-1-ol, has the following structure:
Figure 107116441-A0305-02-0010-10

5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentan-1-ol 5-((2-(6-amino-9 H -purin-9-yl)ethyl)amino)pentan-1-ol

所述NB010，即6-氨基-9-(2-對-甲苯氧基-乙基)-9H-嘌呤-8-硫醇，具有以下化學結構：

Figure 107116441-A0305-02-0010-11
The NB010, namely 6-amino-9-(2-p-tolyloxy-ethyl)-9H-purine-8-thiol, has the following chemical structure:
Figure 107116441-A0305-02-0010-11

6-amino-9-(2-p-tolyloxy-ethyl)-9H-purine-8-thiol 6-amino-9-(2-p-tolyloxy-ethyl)-9H-purine-8-thiol

所述NB011，即4-(9H-嘌呤-6-基)嗎啉(也稱為6-嗎啉-4-基-7H-嘌呤)具有以下化學結構：

Figure 107116441-A0305-02-0010-12
The NB011, namely 4-(9H-purin-6-yl)morpholine (also known as 6-morpholin-4-yl-7H-purine) has the following chemical structure:
Figure 107116441-A0305-02-0010-12

6-morpholin-4-yl-7H-purine。 6-morpholin-4-yl-7H-purine.

(8)、如(1)所述的藥物組合物，其特徵在於，所述類鴉片類藥物是嗎啡。 (8) The pharmaceutical composition according to (1), wherein the opioid drug is morphine.

本發明還提供： The invention also provides:

(9)、一種治療神經性疼痛導致的焦慮和其它疾病相關的焦慮和抑鬱的藥物組合物，所述藥物組合物含有NB001、NB010、或NB011三種化合物中的至少1種。 (9) A pharmaceutical composition for treating anxiety caused by neuropathic pain and anxiety and depression related to other diseases, the pharmaceutical composition containing at least one of the three compounds NB001, NB010, or NB011.

(10)、如(9)所述的藥物組合物，所述NB001、NB010、或NB011三種化合物還包括其可藥用的鹽或溶劑合物。 (10) The pharmaceutical composition according to (9), wherein the three compounds of NB001, NB010, or NB011 further include their pharmaceutically acceptable salts or solvates.

(11)、一種治療慢性內臟疼痛及其相關的焦慮和抑鬱的藥物組合物，所述藥物組合物含有NB001、NB010、或NB011三種化合物中的至少1種。 (11) A pharmaceutical composition for treating chronic visceral pain and related anxiety and depression, the pharmaceutical composition containing at least one of the three compounds NB001, NB010, or NB011.

(12)、一種治療慢性使用嗎啡引起的瘙癢反應的藥物組合物，所述藥物組合物含有NB001、NB010、或NB011三種化合物中的至少1種。 (12) A pharmaceutical composition for treating pruritus caused by chronic use of morphine, the pharmaceutical composition containing at least one of the three compounds NB001, NB010, or NB011.

根據本發明，NB001對於慢性使用嗎啡或其他類鴉片劑化合物的鎮痛具有增強作用，能夠減少耐受性。結合嗎啡，可減少產生類似疼痛緩解水準所需的嗎啡劑量。 According to the present invention, NB001 has an analgesic effect on chronic use of morphine or other opiate compounds, and can reduce tolerance. In combination with morphine, the dose of morphine required to produce a similar level of pain relief can be reduced.

本發明的治療還可用於在用嗎啡治療的患者中增強嗎啡鎮痛效果及/或減少嗎啡耐受性。嗎啡耐受(嗎啡鎮痛作用喪失)可能發生在長時間使用嗎啡的患者身上。在人和動物中，若干天或數周內重複使用嗎啡會導致鎮痛作用減弱。 The treatment of the present invention can also be used to enhance the analgesic effect of morphine and/or reduce morphine tolerance in patients treated with morphine. Morphine tolerance (loss of morphine analgesia) may occur in patients who use morphine for a long time. In humans and animals, repeated use of morphine for several days or weeks can lead to reduced analgesic effects.

本發明的另一方面涉及作為AC1 & 8抑制劑新型化合物NB010和NB011。這是特別重要的，因為AC1和AC8活性都被提出是對類鴉片誘導致敏的關鍵。因此，抑制AC1和AC8活性可能是減少鴉片誘導的耐受性、成癮性和其他作用的理想選擇。本發明中提出，NB001、NB010和NB011的作用原理是因為有效地抑制了AC1和AC8活性，但並非所有抑制AC1和AC8的化合物，都具有本發明的效果。Another aspect of the present invention relates to the novel compounds NB010 and NB011 as AC1 & 8 inhibitors. This is particularly important because both AC1 and AC8 activities have been proposed to be the key to opioid-induced sensitization. Therefore, inhibiting the activity of AC1 and AC8 may be an ideal choice for reducing opiate-induced tolerance, addiction, and other effects. It is proposed in the present invention that the principle of action of NB001, NB010 and NB011 is because they effectively inhibit the activities of AC1 and AC8, but not all compounds that inhibit AC1 and AC8 have the effects of the present invention.

NB010和NB011可用於增加慢性使用嗎啡或其他類鴉片劑化合物的鎮痛作用，以減少耐受性。結合嗎啡，可減少產生類似疼痛緩解水準所需的嗎啡劑量。NB010 and NB011 can be used to increase the analgesic effect of chronic use of morphine or other opiate compounds to reduce tolerance. In combination with morphine, the dose of morphine required to produce a similar level of pain relief can be reduced.

本發明還通過向該人施用有效量的抑制AC1或AC1＆8活性的化合物來治療有需要的人的嗎啡耐受性。 通常這樣一個人在嗎啡治療慢性疼痛時已經形成嗎啡耐受。NB010 / NB011可用於減少長期使用嗎啡和其他鴉片劑引起的加成反應。The present invention also treats the morphine tolerance of a person in need by administering an effective amount of a compound that inhibits the activity of AC1 or AC1&8 to the person. Usually such a person has developed morphine tolerance when morphine treats chronic pain. NB010 / NB011 can be used to reduce the addition reaction caused by long-term use of morphine and other opiates.

在如神經性疼痛，癌症疼痛的慢性疼痛治療中，患者可能會長時間服用止痛藥。 此外，嗎啡治療的副作用可能是瘙癢。 NB010 / NB011可用於減少慢性使用嗎啡引起的瘙癢反應。In the treatment of chronic pain such as neuropathic pain and cancer pain, patients may take painkillers for a long time. In addition, a side effect of morphine treatment may be itching. NB010 / NB011 can be used to reduce the itching response caused by chronic use of morphine.

NB010 / NB011可用於減少受傷引起的情緒焦慮/抑鬱。NB010和NB011可用於減少可能由長期使用類鴉片劑（如嗎啡）或損傷引起的瘙癢。本發明係揭露使用NB010和NB011一般用於抗瘙癢的用途。NB010/NB011 can be used to reduce emotional anxiety/depression caused by injury. NB010 and NB011 can be used to reduce itching that may be caused by long-term use of opiates (such as morphine) or injury. The present invention discloses the general use of NB010 and NB011 for anti-pruritus.

已知AC1和AC8均參與乙醇誘導的敏感性（Maas等，2005），因此，NB010和NB011可用於治療乙醇相關的成癮，敏感性及其他症狀。It is known that both AC1 and AC8 are involved in ethanol-induced sensitivity (Maas et al., 2005). Therefore, NB010 and NB011 can be used to treat alcohol-related addiction, sensitivity and other symptoms.

據報導，AC1和AC8涉及***致敏（DiRocco et al。，2009），因此，發明人提出使用NB010和NB011來治療***長期使用相關的成癮等行為。According to reports, AC1 and AC8 are involved in ***e sensitization (DiRocco et al., 2009). Therefore, the inventors proposed to use NB010 and NB011 to treat long-term ***e-related addiction and other behaviors.

本發明所揭化合物還可採用藥學上可接受的鹽的形式存在。對於在醫藥上使用時，本發明所揭化合物的鹽係指無毒性的“藥學上可接受的鹽”(Ref International J.Pharm.，1986，33，201-217；J.Pharm.Sci.，1997(Jan)，66，1，1)。然而，本領域所屬技術人員熟知將其它鹽用於製備本發明所揭化合物或其藥學上可接受的鹽。代表性的有機或無機酸，包括但不限於，鹽酸、氫溴酸、氫碘酸、高氯酸、硫酸、硝酸、磷酸、乙酸、丙酸、乙醇酸、乳酸、琥珀酸、馬來酸、富馬酸、蘋果酸、酒石酸、枸櫞酸、苯甲酸、扁桃酸、甲磺酸、羥乙磺酸、苯磺酸、草酸、撲酸、2-萘磺酸、對甲苯磺酸、環己烷氨基磺酸、水楊酸、糖精酸或三氟乙酸。代表性有機或無機堿包括但不限於鹼性或陽離子鹽如苄星、氯普魯卡因、膽鹼、二乙醇胺、乙二胺、葡甲胺、普魯卡因、鋁、鈣、鋰、鎂、鉀、鈉和鋅。The compounds disclosed in the present invention can also exist in the form of pharmaceutically acceptable salts. For use in medicine, the salt of the compound disclosed in the present invention refers to a non-toxic "pharmaceutically acceptable salt" (Ref International J. Pharm., 1986, 33, 201-217; J. Pharm. Sci., 1997 (Jan), 66, 1, 1). However, those skilled in the art are familiar with the use of other salts in the preparation of the compounds disclosed in the present invention or their pharmaceutically acceptable salts. Representative organic or inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, perchloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, lactic acid, succinic acid, maleic acid, Fumaric acid, malic acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, isethionic acid, benzenesulfonic acid, oxalic acid, pamoic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, cyclohexane Alkanesulfamic acid, salicylic acid, saccharinic acid or trifluoroacetic acid. Representative organic or inorganic salts include but are not limited to alkaline or cationic salts such as benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, Magnesium, potassium, sodium and zinc.

本發明所揭化合物的前藥係包括在本發明的範圍內。一般而言，此類前藥將是所述化合物的功能性衍生物，很容易在體內轉化為所需化合物。因此，在本發明的治療方法中，術語“給藥”應包括用具體公開的化合物或者用沒有具體公開、但給予患者後在體內轉化為具體化合物的化合物治療各種疾病。The prodrugs of the compounds disclosed in the present invention are included in the scope of the present invention. Generally speaking, such prodrugs will be functional derivatives of the compound, which can be easily converted into the desired compound in the body. Therefore, in the treatment method of the present invention, the term "administration" shall include the treatment of various diseases with a specifically disclosed compound or a compound that is not specifically disclosed but is converted into a specific compound in the body after administration to a patient.

本發明所揭之藥物組合物可以用於腸胃外、口服、，直腸、置換或經皮給藥的形式存在。The pharmaceutical composition disclosed in the present invention can be in the form of parenteral, oral, rectal, replacement or transdermal administration.

因此，它們將可注射溶液、懸浮液或多劑量小瓶之型態呈現，或是普通或包衣片劑、糖衣片、晶片膠囊、凝膠膠囊、丸劑、扁囊劑、粉劑、栓劑或直腸膠囊的形式呈現，用於在極性溶劑中經皮使用，或用於固定使用。Therefore, they present injectable solutions, suspensions or multi-dose vials, or ordinary or coated tablets, sugar-coated tablets, wafer capsules, gel capsules, pills, cachets, powders, suppositories or rectal capsules. It is presented in the form of, for transdermal use in polar solvents, or for fixed use.

適於本發明的賦形劑是纖維素或微晶纖維素衍生物、鹼土金屬碳酸鹽、磷酸鎂、澱粉、改性澱粉和固體形式的乳糖。Excipients suitable for the present invention are cellulose or microcrystalline cellulose derivatives, alkaline earth metal carbonates, magnesium phosphate, starch, modified starch and lactose in solid form.

對於直腸使用來說，可可脂或聚乙二醇硬脂酸酯是優選的賦形劑。For rectal use, cocoa butter or polyethylene glycol stearate are the preferred excipients.

對於腸胃外使用來說，水、水溶液、生理鹽水和等滲溶液是最適當使用的載體。For parenteral use, water, aqueous solutions, physiological saline and isotonic solutions are the most appropriate carriers to use.

對於本領域技術人員來說，本發明活性化合物或其藥物組合物的治療有效劑量將根據所需效果而改變乃為顯而易見者。因此，很容易確定將投予或給藥的最佳劑量，並且，所述劑量將根據所用具體化合物、給藥方式、製劑強度和疾病進展而改變。此外，與具體受治療患者有關的因素，包括患者年齡、體重、飲食和給藥時間，都需要將劑量調整至適當的治療水準。因此，以上劑量是一般情況下的舉例。當然可存在更高或更低劑量的個別情況，這在本發明的範圍之內。It is obvious to those skilled in the art that the therapeutically effective dose of the active compound of the present invention or its pharmaceutical composition will vary according to the desired effect. Therefore, it is easy to determine the optimal dose to be administered or administered, and the dose will vary according to the specific compound used, the mode of administration, the strength of the formulation, and the progression of the disease. In addition, factors related to the specific patient being treated, including the patient's age, weight, diet, and administration time, require the dosage to be adjusted to an appropriate treatment level. Therefore, the above dosage is an example under normal circumstances. Of course, there may be individual cases of higher or lower doses, which are within the scope of the present invention.

每當需要將本發明化合物作為鎮痛藥用於有需要的患者時，都可通過上述組合物和劑量方案，或者通過本領域建立的那些組合物和劑量方案給予本發明化合物。Whenever it is necessary to use the compound of the present invention as an analgesic for a patient in need, the compound of the present invention can be administered through the above-mentioned composition and dosage regimen, or through those compositions and dosage regimens established in the art.

本發明還提供包含一個或多個容器的藥用或獸醫包裝或藥劑盒，所述容器裝有一種或多種本發明所揭藥用和獸醫組合物的成分。任選附隨此類容器的可以是由管理藥物或生物製品的製造、使用或銷售的政府部門發出的通告，所述通告表示得到管理製造、使用或銷售人用藥的部門的批准。The present invention also provides a pharmaceutical or veterinary package or kit comprising one or more containers containing one or more ingredients of the pharmaceutical and veterinary composition disclosed in the present invention. Optionally accompanied by such a container may be a notice issued by a government agency that regulates the manufacture, use, or sale of drugs or biological products, the notice indicating that it has been approved by the agency that regulates the manufacture, use, or sale of human drugs.

通過給予鎮痛有效劑量，本發明化合物係用於治療如人的溫血動物的輕度至重度疼痛。對於普通(70kg)人，在每天約1-4次給藥的方案中，劑量範圍將為約0.1mg-約15,000mg，優選為約50mg-約3500mg，或者更優選為約100mg-約1000mg活性成分；但本領域技術人員清楚本發明活性化合物的治療有效量將隨受治療疼痛的類型而改變。By administering an analgesic effective dose, the compound of the present invention is used to treat mild to severe pain in warm-blooded animals such as humans. For ordinary (70kg) humans, in a regimen of about 1-4 doses per day, the dosage will range from about 0.1 mg to about 15,000 mg, preferably from about 50 mg to about 3500 mg, or more preferably from about 100 mg to about 1000 mg. Ingredients; but those skilled in the art know that the therapeutically effective amount of the active compound of the present invention will vary with the type of pain to be treated.

對於口服給藥，優選提供片劑形式的藥用組合物，所述片劑包含0.01、10.0、50.0、100、150、200、250和500毫克活性成分，根據症狀調節給予受治療患者的劑量。For oral administration, it is preferable to provide a pharmaceutical composition in the form of a tablet containing 0.01, 10.0, 50.0, 100, 150, 200, 250, and 500 mg of the active ingredient, and the dose to be administered to the patient to be treated is adjusted according to the symptoms.

本發明所述的疼痛實例，包括但不限於，炎性疼痛、中樞介導性疼痛、外周介導性疼痛、內臟痛、結構或軟組織損傷相關性疼痛、進行性疾病相關性疼痛、神經性疼痛和急性疼痛(如由急性損傷、外傷或手術導致)和慢性疼痛(如頭痛和神經性疾病、中風後疾病、癌症和偏頭痛導致的頭痛)。Examples of pain in the present invention include, but are not limited to, inflammatory pain, central-mediated pain, peripheral-mediated pain, visceral pain, structural or soft tissue injury-related pain, progressive disease-related pain, and neuropathic pain And acute pain (such as caused by acute injury, trauma, or surgery) and chronic pain (such as headaches and neurological diseases, post-stroke diseases, cancer and migraine headaches).

本發明所揭化合物還可用作免疫抑制劑、抗炎劑、治療和預防神經和精神疾病(如抑鬱症和帕金森病)的藥物、治療泌尿生殖道疾病(如尿失禁和早洩)的藥物、治療藥物濫用和酗酒的藥物、治療胃炎和腹瀉的藥物、心血管藥物和心臟保護劑以及治療呼吸系統疾病的藥物。The compounds disclosed in the present invention can also be used as immunosuppressants, anti-inflammatory agents, drugs for the treatment and prevention of neurological and mental diseases (such as depression and Parkinson's disease), and drugs for the treatment of urinary and reproductive tract diseases (such as urinary incontinence and premature ejaculation) , Drugs for the treatment of drug abuse and alcoholism, drugs for the treatment of gastritis and diarrhea, cardiovascular drugs and cardioprotective agents, and drugs for the treatment of respiratory diseases.

本發明所揭化合物還可用於治療由下列疾病導致的疼痛：骨關節炎、類風濕性關節炎、纖維肌痛、偏頭痛、頭痛、牙痛、燒傷、曬傷、蛇咬傷(特別是毒蛇咬傷)、蜘蛛咬傷、昆蟲螫傷、神經原性膀胱、良性***肥大、間質性膀胱炎、鼻炎、接觸性皮炎/超敏反應、瘙癢、濕疹、咽炎、粘膜炎、腸炎、蜂窩織炎、灼痛、坐骨神經炎、下頜關節神經痛、末梢神經炎、多發性神經炎、殘肢痛、幻肢痛、術後腸梗阻、膽囊炎、***切除術後疼痛綜合征、口腔神經性疼痛、夏科(Charcot)痛、反射***感神經營養不良、格林巴厘綜合征、感覺異常性股痛、口腔燒灼綜合征、皰疹後神經痛、三叉神經痛、叢集性頭痛、偏頭痛、周圍神經病、雙側周圍神經病、糖尿病性神經病變、帶狀皰疹後神經痛、三叉神經痛、視神經炎、發熱後神經炎、遊走性神經炎、節段性神經炎、貢博(Gombault)神經炎、神經元炎、頸臂神經痛、腦神經痛、膝狀節神經痛、舌咽神經痛、偏頭痛性神經痛、特發性神經痛、肋間神經痛、乳腺內神經痛、莫頓(Morton)神經痛、鼻睫神經痛、枕神經痛、紅斑性肢痛病、斯路德神經痛、脾齶(splenopalatine)神經痛、眶上神經痛、翼管神經痛、炎性腸病、腸易激綜合征、竇性頭痛、緊張性頭痛、產程、分娩、經期痙攣和癌症。The compound disclosed in the present invention can also be used to treat pain caused by the following diseases: osteoarthritis, rheumatoid arthritis, fibromyalgia, migraine, headache, toothache, burn, sunburn, snake bite (especially snake bite) , Spider bites, insect stings, neurogenic bladder, benign prostatic hypertrophy, interstitial cystitis, rhinitis, contact dermatitis/hypersensitivity, itching, eczema, pharyngitis, mucositis, enteritis, cellulitis, burning Pain, sciatic neuritis, mandibular neuralgia, peripheral neuritis, polyneuritis, residual limb pain, phantom limb pain, postoperative intestinal obstruction, cholecystitis, post-mastectomy pain syndrome, oral neuropathic pain, Charcot ( Charcot) pain, reflex sympathetic dystrophy, Guillain-Bali syndrome, paresthesia, oral burning syndrome, postherpetic neuralgia, trigeminal neuralgia, cluster headache, migraine, peripheral neuropathy, bilateral peripheral Neuropathy, diabetic neuropathy, post-herpetic neuralgia, trigeminal neuralgia, optic neuritis, post-febrile neuritis, migratory neuritis, segmental neuritis, Gombault neuritis, neuronitis, Cervical-brachial neuralgia, cranial neuralgia, geniculate neuralgia, glossopharyngeal neuralgia, migraine neuralgia, idiopathic neuralgia, intercostal neuralgia, intramammary neuralgia, Morton neuralgia, nose Ciliary neuralgia, occipital neuralgia, erythematous limb pain, Srudd neuralgia, splenopalatine neuralgia, supraorbital neuralgia, vidian neuralgia, inflammatory bowel disease, irritable bowel syndrome, sinus Sexual headaches, tension headaches, labor, childbirth, menstrual cramps, and cancer.

具體實施方式Detailed ways

NB001的中文名稱為5-((2-(6-氨基-9H-嘌呤-9-基)乙基)胺基)戊烷-1-醇，英文名稱為5-((2-(6-amino)-9H-purin-9-yl)ethyl)amino)pentan-1-ol，分子式為C12H20N6O，分子量為264.33(單位)。可購於Sigma公司686301-48-4號產品。 The Chinese name of NB001 is 5-((2-(6-amino-9H-purin-9-yl)ethyl)amino)pentane-1-ol, and the English name is 5-((2-(6-amino )-9H-purin-9-yl)ethyl)amino)pentan-1-ol, the molecular formula is C 12 H 20 N 6 O, and the molecular weight is 264.33 (units). It can be purchased from Sigma Company No. 686301-48-4.

NB010,6-氨基-9-(2-對-甲苯氧基-乙基)-9H-嘌呤-8-硫醇具有以下化學結構：

Figure 107116441-A0305-02-0016-13
NB010, 6-amino-9-(2-p-tolyloxy-ethyl)-9H-purine-8-thiol has the following chemical structure:
Figure 107116441-A0305-02-0016-13

購自Asinex LTD公司的BAS03384號產品。 Product BAS03384 purchased from Asinex LTD.

NB011,4-(9H-嘌呤-6-基)嗎啉(也稱為6-嗎啉-4-基-7H-嘌呤)具有以下化學結構：

Figure 107116441-A0305-02-0017-14
NB011,4-(9H-purin-6-yl)morpholine (also known as 6-morpholin-4-yl-7H-purine) has the following chemical structure:
Figure 107116441-A0305-02-0017-14

購自Maybridge公司JFD02793號產品。 Purchased from Maybridge company JFD02793 product.

在本發明範圍內，是具有所示化學式的化合物的藥學上可接受的鹽係屬於化合物A和化合物B的定義。 Within the scope of the present invention, the pharmaceutically acceptable salt system of the compound having the shown chemical formula belongs to the definition of compound A and compound B.

實施例1.篩選AC1 & 8混合抑制劑 Example 1. Screening of AC1 & 8 mixed inhibitors

進行一組生化篩選試驗，以尋找潛在的AC1和AC8共抑制劑(或稱為AC1和8抑制劑)。在AC1或AC8表達的細胞系中進行cAMP測定，基因啟動(pCREB)測定，以及綜合生理實驗來篩選作用於AC1和AC8上的潛在化合物A和B。發現在100μM下NB010和NB011都產生了AC1以及AC8活性的顯著抑制(見表1)。試驗方法見Wang,H.S.,et al.,Identification of an Adenylyl Cyclase Inhibitor for Treating Neuropathic and Inflammatory Pain.Science Translational Medicine,2011.3(65)。 Conduct a set of biochemical screening tests to find potential AC1 and AC8 co-inhibitors (or AC1 and 8 inhibitors). CAMP assay, gene activation (pCREB) assay, and comprehensive physiological experiments were performed in AC1 or AC8 expressing cell lines to screen potential compounds A and B that act on AC1 and AC8. It was found that both NB010 and NB011 produced significant inhibition of AC1 and AC8 activities at 100 μM (see Table 1). For the test method, see Wang, H.S., et al., Identification of an Adenylyl Cyclase Inhibitor for Treating Neuropathic and Inflammatory Pain. Science Translational Medicine, 2011.3 (65).

Figure 107116441-A0305-02-0017-15
Figure 107116441-A0305-02-0017-15

實施例2. NB010和NB011對焦慮相關的皮質LTP的評估Example 2. Evaluation of NB010 and NB011 on anxiety-related cortical LTP

以前的研究表明，通過重複刺激誘導的突觸LTP需要AC1和AC8活性（Zhuo，2016）。 為了評估NB010和NB011對神經元AC1和AC8活性的影響，本發明人在成年小鼠的ACC皮質切片中記錄了兩種形式的LTP。Previous studies have shown that synaptic LTP induced by repeated stimulation requires AC1 and AC8 activity (Zhuo, 2016). In order to evaluate the effects of NB010 and NB011 on the activity of neurons AC1 and AC8, the inventors recorded two forms of LTP in ACC cortical sections of adult mice.

如前所述，θ刺激刺激誘導持續至少40分鐘的突觸反應的增強（圖1A，n = 8個切片）。 使用NB010（25μM）進行預處理，可以阻斷ACC LTP的誘導（圖1A，n = 5片）。 使用NB011進行預處理也出現了相似的結果（見圖2，n = 6個切片）。 相比之下，同樣劑量（25uM）的非選擇性AC抑制劑SQ22356沒有引起pre-LTP的顯著降低（圖2，n = 5）。作為對比，使用GABApention（25 uM）也沒有阻斷pre-LTP（n = 5）。 ACC最近的一份報告（Chen et al。，2014）也發現GABApention不影響ACC LTP。 有趣的是，forskolin（10uM）導致了pre-LTP的輕微增強（圖2），表明AC的活化對於pre-LTP是關鍵的。 這些發現表明，NB010和NB011都能抑制成年小鼠神經元中的AC1和AC8活性。從而證明了NB010和NB011能夠應該用於治療損傷引起的焦慮。As mentioned earlier, theta stimulation induced an enhancement of synaptic response lasting at least 40 minutes (Figure 1A, n = 8 slices). Pretreatment with NB010 (25μM) can block the induction of ACC LTP (Figure 1A, n = 5 tablets). The use of NB011 for preprocessing also showed similar results (see Figure 2, n = 6 slices). In contrast, the non-selective AC inhibitor SQ22356 at the same dose (25uM) did not cause a significant reduction in pre-LTP (Figure 2, n = 5). For comparison, using GABApention (25 uM) also did not block pre-LTP (n = 5). A recent report by ACC (Chen et al., 2014) also found that GABApention does not affect ACC LTP. Interestingly, forskolin (10uM) caused a slight enhancement of pre-LTP (Figure 2), indicating that AC activation is critical for pre-LTP. These findings indicate that both NB010 and NB011 can inhibit the activity of AC1 and AC8 in adult mouse neurons. This proves that NB010 and NB011 can be used to treat anxiety caused by injury.

因為最近的研究表明，損傷引起的焦慮可能與pre-LTP有關（Koga等，2015; Zhuo，2016）。 因此發明人決定測試NB010和NB011對pre-LTP的影響。 並發現兩種化合物都能抑制pre-LTP（圖1）。具體試驗過程見Koga, K., et al., Coexistence of Two Forms of LTP in ACC Provides a Synaptic Mechanism for the Interactions between Anxiety and Chronic Pain. Neuron, 2015. 85(2): p. 377-389。Because recent studies have shown that anxiety caused by injury may be related to pre-LTP (Koga et al., 2015; Zhuo, 2016). Therefore, the inventor decided to test the influence of NB010 and NB011 on pre-LTP. And found that both compounds can inhibit pre-LTP (Figure 1). For the specific test process, see Koga, K., et al., Coexistence of Two Forms of LTP in ACC Provides a Synaptic Mechanism for the Interactions between Anxiety and Chronic Pain. Neuron, 2015. 85(2): p. 377-389.

圖.1 使用AC1＆8抑制劑NB010在ACC中抑制pre-LTP和post-LTP，其中（A）在對照切片中post-LTP被誘導（實心圓; n =8），而施用NB010（25μM）溶液阻斷了post-LTP的誘導（n = 5）; （B）在對照切片中pre-LTP被誘導（實心圓; n =8），而施用NB010（25μM）溶液阻斷了pre-LTP的誘導（n = 5）。Fig.1 Using AC1&8 inhibitor NB010 to inhibit pre-LTP and post-LTP in ACC, where (A) post-LTP was induced in the control section (closed circle; n=8), and NB010 (25μM) solution was used to block The induction of post-LTP was interrupted (n = 5); (B) pre-LTP was induced in the control section (closed circle; n = 8), while the administration of NB010 (25 μM) solution blocked the induction of pre-LTP ( n = 5).

圖2顯示的是不同試劑抑制AC1/8對ACC中pre-LTP的影響。其中，資料顯示的是誘導pre-LTP 45分鐘時，在最後5分鐘增強。*表示與對照組相比具有顯著差異（其中NB010和NB011導致了減少，而forskolin導致了增加）。Figure 2 shows the effect of different agents inhibiting AC1/8 on pre-LTP in ACC. Among them, the data shows that when pre-LTP is induced for 45 minutes, it is enhanced in the last 5 minutes. * Indicates a significant difference compared with the control group (NB010 and NB011 caused the decrease, and forskolin caused the increase).

實施例3. AC1和AC1＆8抑制劑對嗎啡耐受性的行為影響Example 3. Behavioral influence of AC1 and AC1&8 inhibitors on morphine tolerance

首先，發明人想檢查AC1基因剔除小鼠是否會影響重複注射嗎啡引起的嗎啡耐受。據發明人以前報導，發明人每天注射嗎啡（10mg/ kg，s.c.）7天以測量嗎啡引起的耐受性。發明人發現在AC基因剔除小鼠中，與鹽水處理的野生型小鼠（n = 5-8只小鼠：第7天：對照小鼠，MPE 18±6％，AC1KO，40±8％）相比，嗎啡產生顯著的極大的鎮痛作用; P ＜0.05）。這些結果表明，抑制AC1活性可能有益於減少嗎啡耐受。接下來，發明人使用選擇性AC1抑制劑NB001（5mg / kg，i.p.）來用嗎啡處理野生型小鼠。記錄傷害感受器和尾巴反應延遲。發明人發現NB001聯合應用對慢性嗎啡治療後的應答潛伏期產生顯著影響（p ＜0.005）（圖3）。First, the inventor wanted to examine whether the AC1 gene knockout mice would affect the morphine tolerance caused by repeated injections of morphine. According to previous reports by the inventors, the inventors injected morphine (10 mg/kg, s.c.) every day for 7 days to measure the tolerance caused by morphine. The inventors found that in AC gene knockout mice, wild-type mice treated with saline (n = 5-8 mice: day 7: control mice, MPE 18 ± 6%, AC1KO, 40 ± 8%) In contrast, morphine produced a significant and great analgesic effect; P <0.05). These results indicate that inhibiting AC1 activity may be beneficial in reducing morphine tolerance. Next, the inventors used the selective AC1 inhibitor NB001 (5mg/kg, i.p.) to treat wild-type mice with morphine. Record nociceptor and tail response delays. The inventor found that the combined application of NB001 had a significant impact on the response latency after chronic morphine treatment (p <0.005) (Figure 3).

以前的AC1和AC8雙基因剔除小鼠的研究發現嗎啡耐受性顯著降低（Li et al。，2006）。發明人預計AC1＆8抑制劑也可能產生減少嗎啡耐受性的有益效果。類似地，在嗎啡注射後7天，使用NB010或NB011（5mg/kg，i.p.）的預處理（n = 5-8只小鼠，與每種情況下的對照處理相比p ＜0.05）都產生顯著更大的鎮痛作用。具體試驗方法見Li, S., Lee, M. L., Bruchas, M. R., Chan, G. C., Storm, D. R., and Chavkin, C. (2006). Calmodulin-stimulated adenylyl cyclase gene deletion affects morphine responses. Mol Pharmacol 70, 1742-1749.A previous study of AC1 and AC8 double gene knockout mice found that morphine tolerance was significantly reduced (Li et al., 2006). The inventor predicts that AC1&8 inhibitors may also have the beneficial effect of reducing morphine tolerance. Similarly, 7 days after morphine injection, pretreatment with NB010 or NB011 (5mg/kg, ip) (n = 5-8 mice, p <0.05 compared to the control treatment in each case) produced Significantly greater analgesic effect. For specific test methods, see Li, S., Lee, ML, Bruchas, MR, Chan, GC, Storm, DR, and Chavkin, C. (2006). Calmodulin-stimulated adenylyl cyclase gene deletion affects morphine responses. Mol Pharmacol 70, 1742 -1749.

圖3顯示的是使用AC1抑制劑NB001（5mg / kg）可降低重複注射嗎啡（10mg / kg）誘導的嗎啡耐受性。Figure 3 shows that the use of AC1 inhibitor NB001 (5mg/kg) can reduce the morphine tolerance induced by repeated injections of morphine (10mg/kg).

因此，發明人預期AC1抑制劑NB001以及AC1＆8抑制劑NB010和NB011都可用於增強嗎啡鎮痛效果，從而能夠重複使用嗎啡。Therefore, the inventors expect that both AC1 inhibitor NB001 and AC1&8 inhibitors NB010 and NB011 can be used to enhance the analgesic effect of morphine, so that morphine can be used repeatedly.

如圖4所示，注射嗎啡（10mg/kg）後，NB010和NB011在7天（n=5-7只小鼠）逆轉嗎啡耐受性方面產生了顯著效果。相比之下，100mg/ kg的GABApentin沒有產生顯著效果（n = 6只小鼠）。此外，發明人還測試了非選擇性AC抑制劑SQ22356的作用，發現它在相同劑量下沒有產生顯著的作用，表明AC1/AC8的選擇性抑制是關鍵的。As shown in Figure 4, after injection of morphine (10mg/kg), NB010 and NB011 had a significant effect in reversing morphine tolerance for 7 days (n=5-7 mice). In contrast, GABApentin at 100 mg/kg did not produce a significant effect (n = 6 mice). In addition, the inventors also tested the effect of the non-selective AC inhibitor SQ22356 and found that it did not produce a significant effect at the same dose, indicating that the selective inhibition of AC1/AC8 is key.

為了進一步測試AC1/AC8在嗎啡耐受中的作用，發明人應用了AC啟動劑forskolin，並發現嗎啡在重複施用嗎啡後3天小鼠（n=3只小鼠）更快地產生了耐受性。In order to further test the role of AC1/AC8 in morphine tolerance, the inventors applied the AC activator forskolin and found that the mice (n=3 mice) developed tolerance faster after 3 days of repeated administration of morphine. Sex.

AC1抑制劑NB001（5mg/ kg）和AC1/AC8抑制劑NB010或NB011（5mg/kg）在重複嗎啡注射後7天，重複注射嗎啡（10mg / kg）誘導的嗎啡耐受性降低。GABApentin（100mg/kg）或SQ22356（5mg/kg）沒有產生任何顯著的效果。AC1 inhibitor NB001 (5mg/kg) and AC1/AC8 inhibitor NB010 or NB011 (5mg/kg) 7 days after repeated morphine injection, repeated morphine injection (10mg/kg) induced a decrease in morphine tolerance. GABApentin (100mg/kg) or SQ22356 (5mg/kg) did not produce any significant effects.

為了測試可能的劑量相關效應，發明人還以較低劑量1mg/kg應用NB化合物; 發明人發現NB001/NB010/NB011沒有產生顯著的效果，表明所產生的效果是劑量相關的。In order to test the possible dose-related effects, the inventors also applied the NB compound at a lower dose of 1 mg/kg; the inventors found that NB001/NB010/NB011 did not produce significant effects, indicating that the effects produced are dose-related.

實施例4. 嗎啡的條件性位置偏愛實驗（Conditioned Place Preference, CPP）Example 4. Morphine Conditioned Place Preference (Conditioned Place Preference, CPP)

使用條件性位置偏愛實驗（Conditioned Place Preference, CPP）來驗證CREB突變小鼠對嗎啡的有益效果的反應降低（Walters和Blendy，2001）。接下來在鹽水處理和抑制劑治療的小鼠中誘導嗎啡CPP。發現與鹽水處理的小鼠相比，NB001（5mg / kg，ip）處理的小鼠花費顯著更少的時間探索室的嗎啡配對側，表明AC1在嗎啡的增強性質中起了作用（p＜0.05）。 他們對房間兩側的初始偏好沒有顯著差異。具體試驗方法見Walters CL, Blendy JA (2001) Different requirements for cAMP response element binding protein in positive and negative reinforcing properties of drugs of abuse. J Neurosci 21:9438-9444。Conditioned Place Preference (CPP) was used to verify that CREB mutant mice had a reduced response to the beneficial effects of morphine (Walters and Blendy, 2001). Next, morphine CPP was induced in saline-treated and inhibitor-treated mice. It was found that mice treated with NB001 (5mg/kg, ip) spent significantly less time exploring the morphine paired side of the chamber compared with mice treated with saline, indicating that AC1 played a role in the enhancing properties of morphine (p<0.05 ). There was no significant difference in their initial preferences on both sides of the room. For specific test methods, see Walters CL, Blendy JA (2001) Different requirements for cAMP response element binding protein in positive and negative reinforcing properties of drugs of abuse. J Neurosci 21:9438-9444.

對於嗎啡的條件性位置偏愛實驗，發明人發現NB001使嗎啡誘導偏愛的顯著降低。 NB010和NB011（5mg / kg）都與NB001相似（見圖5）。 相反，相同劑量的非選擇性AC抑制劑SQ22356沒有產生任何抑制作用（見圖5）。 表明AC1 / AC8的抑制是有選擇性的。發明人還測試了GABApention在100mg/kg下的作用。 結果如圖5所示，9天后重複使用嗎啡（10mg/kg）進行誘導位置偏好行為，施用應用AC1抑制劑NB001，AC1/8抑制劑NB010或NB011（5mg/kg）的效果顯著。GABApentin（100mg/kg）或SQ22356（5mg/kg）沒有產生任何顯著的效果。For the conditioned place preference experiment of morphine, the inventors found that NB001 significantly reduced the morphine-induced preference. Both NB010 and NB011 (5mg/kg) are similar to NB001 (see Figure 5). In contrast, the same dose of non-selective AC inhibitor SQ22356 did not produce any inhibitory effect (see Figure 5). It shows that the inhibition of AC1/AC8 is selective. The inventor also tested the effect of GABApention at 100 mg/kg. The results are shown in Figure 5. After 9 days, repeated use of morphine (10mg/kg) to induce position preference behavior, the application of AC1 inhibitor NB001, AC1/8 inhibitor NB010 or NB011 (5mg/kg) has a significant effect. GABApentin (100mg/kg) or SQ22356 (5mg/kg) did not produce any significant effects.

另外，試驗結果還顯示（圖未示出），當NB001 / NB010 / NB011的劑量降低到1mg/kg時，沒有產生預期的效果，因此NB001 / NB010 / NB011的作用具有劑量依賴性（n=3-5只小鼠）。In addition, the test results also show (not shown in the figure) that when the dose of NB001 / NB010 / NB011 is reduced to 1 mg/kg, the expected effect is not produced, so the effect of NB001 / NB010 / NB011 is dose-dependent (n=3 -5 mice).

Forskolin（5mg / kg）沒有產生任何抑制作用，從結果看還略有增加選擇時間（n=3只小鼠）。Forskolin (5mg/kg) did not produce any inhibitory effect, and from the results, it slightly increased the selection time (n=3 mice).

實施例5. 焦慮行為測試Example 5. Anxiety Behavior Test

如前所述，發明人發現神經損傷在EPM測試中引起焦慮行為（Koga等，2015;見圖6）。 因此發明人檢查NB010或NB011是否可以減輕焦慮等行為，因為這兩種抑制劑都阻止焦慮相關的pre-LTP。 如預期的那樣，NB010或NB011（5mg / kg）產生由神經損傷引起的焦慮反應顯著降低（每種情況下P ＜0.05，每種試劑n = 6只小鼠）（參見圖6）。As mentioned earlier, the inventors found that nerve damage caused anxiety in EPM testing (Koga et al., 2015; see Figure 6). Therefore, the inventors examined whether NB010 or NB011 can reduce anxiety and other behaviors, because both inhibitors prevent anxiety-related pre-LTP. As expected, NB010 or NB011 (5mg/kg) produced a significant reduction in the anxiety response caused by nerve damage (P<0.05 in each case, n=6 mice per reagent) (see Figure 6).

在較低劑量（1mg / kg）下，NB010（參見圖6）沒有顯著降低。使用1mg / kg的 NB011得到的結果也類似。 GABApentin（100mg / kg，i.p.）和SQ22356（5mg / kg）均未顯著減輕行為焦慮（圖3）。而使用 forskolin（5mg / kg）沒有進一步增強焦慮，這可能是由於產生了飽和效應（圖6）At the lower dose (1mg/kg), NB010 (see Figure 6) did not decrease significantly. The results obtained using 1mg/kg of NB011 are similar. Neither GABApentin (100mg/kg, i.p.) nor SQ22356 (5mg/kg) significantly reduced behavioral anxiety (Figure 3). The use of forskolin (5mg/kg) did not further enhance anxiety, which may be due to the saturation effect (Figure 6)

實驗方法詳見Koga, K., et al., Coexistence of Two Forms of LTP in ACC Provides a Synaptic Mechanism for the Interactions between Anxiety and Chronic Pain. Neuron, 2015. 85(2): p. 377-389。The experimental method is detailed in Koga, K., et al., Coexistence of Two Forms of LTP in ACC Provides a Synaptic Mechanism for the Interactions between Anxiety and Chronic Pain. Neuron, 2015. 85(2): p. 377-389.

圖6顯示的是不同試劑對神經損傷引起的焦慮的影響。其中使用AC1＆8抑制劑NB010 / NB011（5mg / kg，i.p.）降低了由神經損傷引起的焦慮狀態（損傷後7天）。 在1mg / kg的低劑量下，NB010和NB011均不影響焦慮。 GABApentin（100mg / kg）或SQ22356（5 mg / kg）的使用無顯著療效。 本實施例是使用EPM測試來評估焦慮狀態。Figure 6 shows the effects of different agents on anxiety caused by nerve damage. Among them, the use of AC1&8 inhibitor NB010/NB011 (5mg/kg, i.p.) reduced the anxiety state caused by nerve injury (7 days after injury). At the low dose of 1mg/kg, neither NB010 nor NB011 affect anxiety. The use of GABApentin (100mg/kg) or SQ22356 (5 mg/kg) has no significant effect. This embodiment uses the EPM test to assess the anxiety state.

瘙癢的反應Itching response

眾所周知，瘙癢常常是由各種疾病引起的，包括慢性使用類鴉片類物質。 為了評估抑制AC1和AC8可能減少瘙癢反應，發明人評估了NB010和NB011對注射化合物A的行為瘙癢反應的影響。具體試驗方法參考美國專利公開號US20110098295 A1，結果發現發現通過皮內注射化合物48/80（100μg/50μl）誘導的總劃痕反應，與野生型小鼠（n = 7）相比，使用NB010或NB011預處理（n = 6只小鼠）注射的小鼠其總劃痕反應顯著減少（圖7）。 GABApention（100mg / kg）的應用沒有引起瘙癢反應的顯著降低（圖7）。 SQ22356在測試的劑量（5mg / kg，腹膜內）下也沒有顯著降低。 相比之下，forskolin（5 mg / kg）的應用顯著增強了行為瘙癢反應（圖7）。As we all know, itching is often caused by various diseases, including chronic use of opioids. In order to evaluate that inhibiting AC1 and AC8 may reduce the pruritus response, the inventors evaluated the effects of NB010 and NB011 on the behavioral pruritus response of injection of compound A. For the specific test method, refer to US Patent Publication No. US20110098295 A1, and it was found that the total scratch response induced by intradermal injection of compound 48/80 (100μg/50μl) was compared with wild-type mice (n=7) using NB010 or Mice injected with NB011 pretreatment (n = 6 mice) had a significant reduction in total scratch response (Figure 7). The application of GABApention (100mg/kg) did not cause a significant reduction in the itching response (Figure 7). SQ22356 also did not decrease significantly at the tested dose (5mg/kg, intraperitoneal). In contrast, the application of forskolin (5 mg/kg) significantly enhanced the behavioral itching response (Figure 7).

圖7顯示了影響cAMP途徑的不同試劑對小鼠行為瘙癢反應的影響。NB010或NB011的應用顯著降低了組胺釋放化合物40/80在成年小鼠中引起的瘙癢反應。 相比之下，GABApentin或SQ22356沒有產生任何顯著的影響。 Forskolin是AC啟動劑，顯著增強了瘙癢反應。 *與對照相比P ＜0.05。Figure 7 shows the effect of different agents that affect the cAMP pathway on behavioral pruritus in mice. The application of NB010 or NB011 significantly reduced the itching response caused by the histamine releasing compound 40/80 in adult mice. In contrast, GABApentin or SQ22356 did not produce any significant effects. Forskolin is an AC initiator, which significantly enhances the itching response. *P<0.05 compared to the control.

試驗動物Test animal

AC1和AC8 KO小鼠參見Wei F, Qui CS, Kim SJ, et al. Genetic elimination of behavioral sensitization in mice lacking calmodulin-stimulated adenylyl cyclases. Neuron. 2002; 36(4): 713-726，並在C57B1/6背景上繁殖了數代（F8-F12）。對照野生型（WT）小鼠是來自Charles River的成年雄性（8-12周齡）C57B1/6小鼠。在實驗結束時，動物被過量吸入麻醉劑（氟烷）處理。動物在光照與黑暗12h：12h的迴圈中飼養，食物和水按照常規提供。AC1 and AC8 KO mice see Wei F, Qui CS, Kim SJ, et al. Genetic elimination of behavioral sensitization in mice lacking calmodulin-stimulated adenylyl cyclases. Neuron. 2002; 36(4): 713-726, and in C57B1/ Several generations (F8-F12) were bred on the background. Control wild-type (WT) mice are adult male (8-12 weeks old) C57B1/6 mice from Charles River. At the end of the experiment, the animals were treated with an overdose of inhaled anesthetic (halothane). Animals are raised in a 12h:12h circle of light and darkness, and food and water are provided as usual.

AC8和AC1新型抑制劑的生化篩選試驗Biochemical Screening Test of New Type Inhibitors of AC8 and AC1

對於AC8表達載體pcDNA3-AC1轉染，將HEK293細胞以每平板1×106的密度鋪板在60mm直徑的培養皿上，生長過夜，並通過Lipofectamine 2000（Invitrogen）轉染pcDNA3-AC1（每片0.8μgDNA） ）。在含有0.8mg / ml G418（Invitrogen，CA）的培養基中選擇穩定的轉染克隆並保持在該培養基中。對於HEK293細胞中其他AC同工型的暫態表達，將HEK293細胞接種在96孔組織培養皿中，分別用AC5質粒轉染，轉染後48 h進行實驗。For the AC8 expression vector pcDNA3-AC1 transfection, HEK293 cells were plated on a 60mm diameter petri dish at a density of 1×106 per plate, grown overnight, and transfected with pcDNA3-AC1 (0.8μg DNA per plate) by Lipofectamine 2000 (Invitrogen) ) ). Select stable transfected clones in a medium containing 0.8 mg/ml G418 (Invitrogen, CA) and keep them in this medium. For the transient expression of other AC isoforms in HEK293 cells, HEK293 cells were seeded in 96-well tissue culture dishes and transfected with AC5 plasmid respectively, and the experiment was performed 48 h after transfection.

收集表達ACs的HEK293細胞，並在不同處理後在0.1M HCl中裂解。使用直接cAMP酶免疫測定試劑盒（Assay Designs，MI）進行直接cAMP測量，並通過酶標儀在405nm測量光密度值。通過向培養物中加入1mM 3-異丁基-1-甲基黃嘌呤（Sigma，MO）來抑制磷酸二酯酶。HEK293 cells expressing ACs were collected and lysed in 0.1M HCl after different treatments. Direct cAMP measurement was performed using the direct cAMP enzyme immunoassay kit (Assay Designs, MI), and the optical density value was measured at 405nm by a microplate reader. Phosphodiesterase was inhibited by adding 1 mM 3-isobutyl-1-methylxanthine (Sigma, MO) to the culture.

CRE螢光素酶報告基因檢測CRE luciferase reporter gene detection

在不存在抗生素的情況下將HEK293細胞傳代培養到96孔板中並生長過夜，並用Lipofectamine 2000試劑用pGL3-CRE螢火蟲螢光素酶和pGL3-CMV-腎上腺素螢光素酶構建體（每孔0.25μgDNA）轉染。將轉染的細胞孵育過夜，並將培養基更換為含有10％胎牛血清的DMEM。 48小時後，每種化學物質的濃度下，用10μM forskolin，10μMA23187和2mM CaCl2或10μM forskolin，10μMA23187和2mM CaCl2的組合處理細胞。在6小時結束時，收穫培養細胞，並通過雙螢光素酶報告分析系統（Promega）測定螢光素酶活性。使用SIRIUS發光計測量相對光單位。In the absence of antibiotics, HEK293 cells were subcultured into 96-well plates and grown overnight, and used Lipofectamine 2000 reagent with pGL3-CRE firefly luciferase and pGL3-CMV-adrenaline luciferase constructs (each 0.25μg DNA) for transfection. The transfected cells were incubated overnight, and the medium was changed to DMEM containing 10% fetal bovine serum. After 48 hours, at the concentration of each chemical substance, the cells were treated with a combination of 10 μM forskolin, 10 μMA23187 and 2mM CaCl2 or 10 μM forskolin, 10 μMA23187 and 2mM CaCl2. At the end of 6 hours, the cultured cells were harvested, and the luciferase activity was measured by the dual luciferase reporter analysis system (Promega). A SIRIUS luminometer was used to measure relative light units.

全細胞膜片鉗記錄Whole cell patch clamp recording

使用標準方法製備ACC水準的冠狀腦切片（300μm）（Wu et al。，2005）。將切片轉移到含氧（95％O 2和5％CO 2）人造腦脊液（ACSF）的浸沒回收室中，（所述人造腦脊液含有mM：124 NaCl，2.5 KCl，2 CaCl2，1 MgSO4，25 NaHCO3，1 NaH2PO4，10葡萄糖）在室溫下至少放置1小時。在配備有用於視覺化的紅外DIC光學器件的BX51W1顯微鏡的臺上的記錄室中進行實驗。使用Axon 200B放大器（Axon Instruments，CA）從II/III神經元記錄興奮性突觸後電流（EPSCs），並通過置於ACC層V的雙極鎢刺激電極遞送刺激物。通過0.05Hz的重複刺激誘導α-氨基-3-羥基-5-甲基異惡唑-4-丙酸（AMPA）受體介導的EPSCs，並且在AP5（50μM）存在下將神經元電壓鉗位元在-70mV。填充記錄移液器（3-5MΩ）的內部溶液為（mM）：145 K-葡萄糖酸鹽，5 NaCl，1 MgCl2，0.2 EGTA，10 HEPES，2 Mg-ATP，0.1 Na3-GTP，10 磷酸肌酸二鈉的溶液，並用KOH調節PH至7。使用內溶液（mM：140 銫甲烷磺酸鈉，5 NaCl，0.5 EGTA，10 HEPES，2 MgATP，0.1 Na3-GTP，0.1精胺，2 QX-314溴化物 和10 磷酸肌酸二鈉（用CsOH調節至pH 7.2） 整流AMPA受體介導的傳遞實驗。對於微型EPSC（mEPSC）記錄，在灌注溶液中加入0.5μMTTX。在所有實驗中，總是存在微量毒素（100μM）以阻斷GABAA受體介導的抑制性突觸電流。通路阻力為15-30MΩ，並在整個實驗過程中進行監測。在實驗過程中，如果通路阻力變化超過15％，則放棄資料。 資料以1kHz過濾，並以10kHz數位化。Use standard methods to prepare ACC-level coronal brain slices (300μm) (Wu et al., 2005). Transfer the slices to an immersion recovery chamber containing oxygen (95% O 2 and 5% CO 2) artificial cerebrospinal fluid (ACSF), (the artificial cerebrospinal fluid contains mM: 124 NaCl, 2.5 KCl, 2 CaCl2, 1 MgSO4, 25 NaHCO3 , 1 NaH2PO4, 10 glucose) at room temperature for at least 1 hour. Experiments were performed in a recording room on the stage of a BX51W1 microscope equipped with infrared DIC optics for visualization. Axon 200B amplifier (Axon Instruments, CA) was used to record excitatory postsynaptic currents (EPSCs) from II/III neurons, and the stimulus was delivered through bipolar tungsten stimulation electrodes placed in ACC layer V. Repetitive stimulation at 0.05 Hz induces α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated EPSCs, and voltage clamping of neurons in the presence of AP5 (50μM) The bit is at -70mV. The internal solution of the filling and recording pipette (3-5MΩ) is (mM): 145 K-gluconate, 5 NaCl, 1 MgCl2, 0.2 EGTA, 10 HEPES, 2 Mg-ATP, 0.1 Na3-GTP, 10 phosphonic acid A solution of disodium acid, and adjust the pH to 7 with KOH. Use internal solution (mM: 140 cesium methanesulfonate sodium, 5 NaCl, 0.5 EGTA, 10 HEPES, 2 MgATP, 0.1 Na3-GTP, 0.1 spermine, 2 QX-314 bromide and 10 creatine disodium phosphate (using CsOH) Adjust to pH 7.2) rectify AMPA receptor-mediated delivery experiments. For micro EPSC (mEPSC) recording, add 0.5 μMTTX to the perfusion solution. In all experiments, there is always a trace of toxin (100 μM) to block GABAA receptors Mediated inhibitory synaptic current. The pathway resistance is 15-30MΩ and is monitored throughout the experiment. During the experiment, if the pathway resistance changes by more than 15%, the data is discarded. The data is filtered at 1kHz and at 10kHz Digitize.

條件性位置偏愛實驗Conditioned place preference experiment

使用了具有兩個截然不同的環境（不同的牆壁，地板和氣味）的房間（MED-associates，St. Albans，VT）。在測試的第一天，允許動物自由地探索腔室的兩側30分鐘，並且使用資料將動物分成幾乎相等偏倚的組。在接下來的八天中，每個動物在室的不同側面上交替天數給予10mg / kg嗎啡或等量體積的鹽水。動物被限制在室的特定側30分鐘。調理後，用生理鹽水注射所有動物，使其自由探索腔室兩側30分鐘。位置偏愛被定義為與以前相比，調節後嗎啡配對側的時間增加。A room (MED-associates, St. Albans, VT) with two distinct environments (different walls, floors and smells) was used. On the first day of the test, the animals were allowed to freely explore both sides of the chamber for 30 minutes, and the data were used to divide the animals into groups of almost equal bias. For the next eight days, each animal was given 10 mg/kg morphine or an equal volume of saline on alternate days on different sides of the chamber. The animal was confined to a specific side of the chamber for 30 minutes. After conditioning, all animals were injected with saline and allowed to freely explore both sides of the chamber for 30 minutes. Position preference is defined as an increase in time on the morphine paired side after adjustment compared to before.

神經性疼痛模型Neuropathic pain model

如前所述，通過連接腓總神經（CPN）誘導神經性疼痛模型（Li等，2010）。簡言之，通過腹膜內注射***（0.16mg / kg; Bimeda-MTC）和賽拉嗪（0.01mg / kg;拜耳）的混合鹽水麻醉小鼠。在幾乎橫向運行的肌肉的前組和後組之間可見CPN。將慢性CPN與鉻腸縫線5-0（Ethicon）緩慢連接，直到足部的背屈肌收縮可見為數位的抽搐。術後第3天，第7天，第14天進行機械異常性疼痛測試。實驗進行盲試，不同個體負責手術和小鼠機械靈敏度的測量。As mentioned earlier, the neuropathic pain model is induced by connecting the common peroneal nerve (CPN) (Li et al., 2010). Briefly, mice were anesthetized by intraperitoneal injection of a mixed saline solution of ketamine (0.16 mg/kg; Bimeda-MTC) and xylazine (0.01 mg/kg; Bayer). CPN can be seen between the front and back groups of muscles that run almost laterally. Slowly connect chronic CPN with chrome intestinal suture 5-0 (Ethicon) until the contraction of the dorsiflexor muscle of the foot can be seen as a digital twitch. The mechanical allodynia test was performed on the 3rd, 7th, and 14th day after operation. The experiment was conducted blindly, and different individuals were responsible for the measurement of surgery and mechanical sensitivity of mice.

機械異常性疼痛試驗Mechanical allodynia test

將小鼠置於圓形容器中，並在測試前適應30分鐘。基於後爪對von Frey絲（Stoelting）應用於彎曲點的回應性來評估機械異常性疼痛。積極的回應包括舔、咬和突然撤回後爪。進行實驗以表徵閾值刺激。發現來自1.65絲的機械壓力（力，0.008g）在幼稚的小鼠中是無害的。然後將該細絲用於測試機械異常性疼痛。The mice are placed in a circular container and acclimatized for 30 minutes before testing. The mechanical allodynia was evaluated based on the responsiveness of the hind paw to von Frey wire (Stoelting) applied to the bending point. Positive responses include licking, biting, and sudden withdrawal of the hind paw. Experiments are performed to characterize the threshold stimulus. The mechanical pressure (force, 0.008 g) from 1.65 silk was found to be harmless in naive mice. The filament is then used to test for mechanical allodynia.

無no

圖1 係AC1＆8抑制劑NB010在ACC中抑制pre-LTP和post-LTP的效果。 圖2 係NB010和NB011對焦慮相關的皮質LTP之評估結果。Figure 1 The effect of AC1&8 inhibitor NB010 in inhibiting pre-LTP and post-LTP in ACC. Figure 2 shows the evaluation results of NB010 and NB011 on anxiety-related cortical LTP.

圖3 係嗎啡耐受性試驗結果。 Figure 3 shows the results of the morphine tolerance test.

圖4 係嗎啡耐受性試驗結果。 Figure 4 shows the results of the morphine tolerance test.

圖5 嗎啡的條件性位置偏愛實驗結果。 Figure 5 Experimental results of conditioned place preference of morphine.

圖6 係不同試劑對神經損傷引起的焦慮的影響。 Figure 6 shows the effects of different reagents on anxiety caused by nerve damage.

圖7 係瘙癢反應試驗結果。 Figure 7 shows the results of the pruritus reaction test.

Figure 107116441-A0101-11-0001-1
Figure 107116441-A0101-11-0001-1

Claims (3)

一種藥物組合物在製備治療神經性疼痛導致的焦慮和其它疾病相關的焦慮和抑鬱的藥物中的用途，所述藥物組合物含有NB001、NB010、NB011、及其可藥用的鹽中的至少一種，所述NB001，具有以下結構：
Figure 107116441-A0305-02-0026-16
 所述NB010，具有以下化學結構：
Figure 107116441-A0305-02-0026-17
 所述NB011，具有以下化學結構：
Figure 107116441-A0305-02-0026-18
 Use of a pharmaceutical composition in the preparation of a medicament for treating anxiety caused by neuropathic pain and other disease-related anxiety and depression, the pharmaceutical composition containing at least one of NB001, NB010, NB011, and pharmaceutically acceptable salts thereof , The NB001 has the following structure:
Figure 107116441-A0305-02-0026-16
 The NB010 has the following chemical structure:
Figure 107116441-A0305-02-0026-17
 The NB011 has the following chemical structure:
Figure 107116441-A0305-02-0026-18
 一種藥物組合物在製備治療慢性內臟疼痛及其相關的焦慮和抑鬱的藥物中的用途，所述藥物組合物含有NB001、NB010、NB011、及其可藥用的鹽中的至少一種，所述NB001，具有以下結構：
Figure 107116441-A0305-02-0027-19
 所述NB010，具有以下化學結構：
Figure 107116441-A0305-02-0027-20
 所述NB011，具有以下化學結構：
Figure 107116441-A0305-02-0027-21
 Use of a pharmaceutical composition in the preparation of a medicament for treating chronic visceral pain and related anxiety and depression. The pharmaceutical composition contains at least one of NB001, NB010, NB011, and pharmaceutically acceptable salts thereof, and the NB001 , Has the following structure:
Figure 107116441-A0305-02-0027-19
 The NB010 has the following chemical structure:
Figure 107116441-A0305-02-0027-20
 The NB011 has the following chemical structure:
Figure 107116441-A0305-02-0027-21
 一種藥物組合物在製備治療慢性使用嗎啡引起的瘙癢反應的藥物中的用途，所述藥物組合物含有選自NB001、NB010、NB011、及其可藥用的鹽中的至少一種，所述NB001，具有以下結構：
Figure 107116441-A0305-02-0028-22
 所述NB010，具有以下化學結構：
Figure 107116441-A0305-02-0028-23
 所述NB011，具有以下化學結構：
Figure 107116441-A0305-02-0028-24
 Use of a pharmaceutical composition in the preparation of a medicament for treating pruritus caused by chronic use of morphine, the pharmaceutical composition containing at least one selected from the group consisting of NB001, NB010, NB011, and pharmaceutically acceptable salts thereof, said NB001, Has the following structure:
Figure 107116441-A0305-02-0028-22
 The NB010 has the following chemical structure:
Figure 107116441-A0305-02-0028-23
 The NB011 has the following chemical structure:
Figure 107116441-A0305-02-0028-24
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